Journal of Air Pollution and Health (Winter 2016); 1(1): 21-26
Original Article
Available online at http://japh.tums.ac.ir
DETERMINATION OF URINARY CONCENTRATIONS OF
ORGANIC SOLVENT IN URBAN WORKERS
Reza Ahmadkhaniha1, Masud Yunesian2,3, Maryam Zare Jeddi2,4, Noushin Rastkari3*
Department of Human Ecology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
Department of Environmental Health Engineering, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
3
Center for Air Pollution Research (CAPR), Institute for Environmental Research (IER), Tehran University of Medical Sciences, Tehran, Iran
4
Institutes for Environmental Research (IER), Tehran University of Medical Sciences, Tehran, Iran
1
2
A RT I C L E I N F O R M AT I O N
ABSTRACT
Article Chronology:
Introduction: Concerns about indoor air quality have drawn researcher’s
attention in the last years. This becomes more important with knowledge of
that 90% of people’s daily times are spent inside the home and workplaces.
Solvents are an example of prevalent hazard chemical, which are lessstudied comparing pesticides or metals. Chlorinated solvents such as carbon
tetrachloride, chloroform, and dichloromethane constitute an important class
of solvent, which applies for a variety of consumer and industrial cleaning
purposes especially in the laboratory. Mentioned components represent
various side effects and carcinogenic implication that could adversely affect
workers exposed to solvents.
Materials and Methods: In the present study the excretion of urinary carbon
tetrachloride, chloroform, and dichloromethane were evaluated as biomarkers
of exposure to chlorinated solvents. With this aim, forty chemistry laboratory
technicians from several universities in Tehran and forty occupationally nonexposed persons were investigated. Spot urine samples were obtained prior
to and at the end of the work shift from each subject. The urinary levels of
chlorinated solvents were determined by using headspace gas chromatography
and mass spectrometry detection.
Results: The mean concentrations of chloroform and dichloromethane in
chemistry laboratory technicians were significantly greater than the control
groups. Although the mean levels of carbon tetrachloride before the work
shift in technicians were higher than the occupationally non-exposed group, a
statistically significant difference could not be observed (Pvalue= 0.324).
Conclusion: The results showed that the laboratory technicians are one of the
most exposed groups among occupationally exposed people with the main
route of exposure through inhalation.
Received 25 July 2015
Revised 22 August 2015
Accepted 21 October 2015
Keywords:
Chloroform, dichloromethane, chlorinated
solvents, Biomarkers
CORRESPONDING AUTHOR:
[email protected]
Tel.: (+98 21) 88978395
Fax: (+98 21) 88978398
INTRODUCTION
Recently, there has been wide concern about the
effects of indoor air quality on human health.
This is frequently, often due to the fact that
almost 90% of people’s every day time is being
spent inside either workplaces and home [1].
Occupational environments have always been
a source of chemical exposure to workers from
point and non-point sources [2]. However, the
indoor air quality has not taken into consideration
as it should have been. Furthermore, the places
Please cite this article as: Ahmadkhaniha R, Yunesian M, Zare Jeddi M, Rastkari N. Determination of urinary concentrations of
organic solvent in urban workers. Journal of Air Pollution and Health. 2016; 1(1):21-26.
22
R. Ahmadkhaniha et al., Determination of urinary concentrations of …
which people are in direct contact with chemical
compounds, including chemical laboratory could
pose human at risk [3]. Therefore, this issue
should be paid more attention; otherwise, people
may suffer from the poor indoor working place
air quality [4]. Situations of human exposure to
chemical hazards include during transportation,
distribution and application of organic solvents
into the immediate environment [5]. Although
long considered as possible human disease risk
factors, solvents have received somewhat less
attention than pesticides or metals despite the
prevalence solvent used in many workplaces
[6]. Solvents are classified by their chemical
characteristics, organic or inorganic, and also
by the chemical composition, such as chlorine
substitution [7]. Exposure can occur through
inhalation due to volatilization of the solvent,
or dermal uptake, or ingestion, depending on
exposure source and chemical composition.
Chlorinated solvents such as carbon tetrachloride
(CTC), chloroform, and dichloromethane (DCM)
have been used for a variety of consumer and
industrial cleaning purposes due to their ability
to dissolve organic substances [8]. These
three chlorinated solvents are among the most
widely used in this solvent group. Most of
chlorinated solvents are common solvents in the
laboratory because they are relatively unreactive
and miscible with most organic liquids, and
conveniently volatile [9]. The prevalence use
of DCM, CTC and chloroform and on the other
hand the resultant potential for exposure to them
representing a concern to public health. Many
various side effects including toxicity to the liver,
kidney, lungs, neurotoxicological effects, and
carcinogenic effects have been reported in the
literature [8, 10-13]. These chlorinated solvents
(DCM, CTC, and chloroform) are classified in
the 2B class (“possible” human carcinogen) by
the International Agency for Research on Cancer
[14].
In recent decades, several studies have
demonstrated the ability of urinary chemicals
to be used as biomarkers of exposure [15-22].
Recent studies showed that the determination
http://japh.tums.ac.ir
of unmetabolized solvents in urine provides a
highly sensitive and specific index of exposure
to chlorinated solvent [15, 17-22]. Chloroform,
CTC and DCM can be eliminated unchanged
in exhaled air and urine. For the assessment of
occupational exposure to the chlorinated solvents
determination of the unmetabolized compounds
or their metabolites in urine and blood were
suggested [23-28]. Therefore, analysis of the
concentration of unmetabolized compounds in
urine detected after the work shift seems to give
the most reliable estimation of exposure, so in
this study the unmetabolized chloroform, CTC
and DCM were selected as suitable biomarkers
of routine solvents exposure. The aim of this
study was to determine the exposure levels
of chloroform, CTC and DCM for laboratory
technicians during routine work shift, by
biological monitoring.
MATERIALS AND METHODS
Study population
Eighty healthy men from the city of Tehran, Iran,
were enrolled in this study. The study population
included 40 chemistry laboratory technicians from
several universities in Tehran as an occupationally
exposed group and for the measurement of any
background levels originating from other sources
such as ambient air, urine samples were collected
from 40 occupationally non-exposed persons in
the same organization acted as referents. All of
the subjects were men between 27 and 43 (mean)
29years old and none of them were smokers. In
order to dermal protection, it was requested from
laboratory technicians to wear plastic gloves
during work shift.
Sampling
The exposure measurements were carried out in
September 2012. Urine samples were collected
at the beginning and the end of the shift. Urine
samples were stored in a cooling box until
transferred to the laboratory, where they were
divided into several fractions and frozen at -20°C
until analysis.
Journal of Air Pollution and Health (Winter 2016); 1(1): 21-26
Analysis of urine samples
Head space solid-phase microextraction (HSSPME) appears to be a solvent- free extraction
technique as an attractive alternative to most
of
the conventional sampling techniques [29].
?????
This technique is based on the distribution
of the analytes to a fused-silica fiber coated
with a stationary phase. For the HS- SPME
holder and fibers were used. The fibers were
determinations,
holderthan
and fibers
conditioned ata manual
20° C SPME
a higher
the
were
used.
The
fibers
were
conditioned
at
20°C
desorption temperature (270° C). Two blank
higher
than were
the desorption
injections
performedtemperature
before the (270°C).
actual
Two
blank
injections
were
performed
before
the
analysis. Between uses, fibers were kept sealed
actual
analysis. air
Between
uses, fibers
kept
from ambient
by piercing
the tipwere
of the
SPMEfrom
needle
into a small
of septum
sealed
ambient
air bypiece
piercing
the tiptoof
prevent
accidental
contamination.
The
HS-to
the SPME needle into a small piece of septum
SPME accidental
parameters
were determined
by
prevent
contamination.
The HS-SPME
experiments
in
which
some
parameters
kept
parameters were determined by experiments in
constant
and parameters
the remaining
was modified
which
some
keptone
constant
and the
to find an optimum condition [30]. Urine
remaining one was modified to find an optimum
samples were collected in polyethylene bottles.
condition [30]. Urine samples were collected
An amount of 2 ml of urine was immediately
intransferred
polyethylene
of 2 vials
ml of
intobottles.
the 10Anmlamount
headspace
urine
was immediately
transferred
the 10-ml
containing
1 g NaCl to
saturate into
the aqueous
headspace
vials containing
NaCl to
saturate
solution. Then,
0.2 ml of1ginternal
standard
the
aqueous
solution.
Then
0.2
ml
of
(hexane in water, 160 ug/l) was added.internal
The
standard
(hexane
water,
added.
vials were
sealed in
using
the160
capsug/l)
withwas
a Teflon
membrane.
The
vial was
placed
in awith
water
bath
The
vials were
sealed
using
the caps
a Teflon
maintained
at
60
±
0.1°
C
for
15
minutes
to
membrane. The vial was placed in a water bath
establish phase
equilibrium.
and
maintained
at 60±0.1
°C for 15 The
min tovial
establish
SPME
holder
were
clamped
into
a
stand
that
phase equilibrium. The vial and SPME holder
allowed
the vial
immersed
in the the
water
were
clamped
intotoa be
stand
that allowed
vial
bath only up to the level of the liquid in the
to be immersed in the water bath only up to the
vial. Next, the SPME needle was exposed to
level of the liquid in the vial. Next, the SPME
headspace so as to locate the tip of the exposed
needle
was exposed to
so as
fiber approximately
0.5headspace
cm from the
toptooflocate
the
the
tip ofHeadspace
the exposedadsorption
fiber approximately
cm
liquid.
time was0.535
from
the top
the was
liquid.
Headspace
minutes.
Theoffiber
then
retracted, adsorption
removed
time
was
35
min.
The
fiber
was then retracted,
from the vial, and placed immediately
into the
removed
from
andAfter
placed
immediately
injector of
the the
GC vial,
system.
that,
the fiber
was the collected
in the
into
injector of and
the GCinserted
system. After
that,
chromatograph injector. Desorption time in the
23
the fiber was collected and inserted in the
chromatograph injector. Desorption time in the
injector was 4.5 min, and the splitter was opened
after 3 min. Determinations were performed by
means of gas chromatography (Agilent GC/MS
6890/5973 detector; HP-5, 30 m × 0.25 mm × 1
µm). The temperatures were as follows: injector
temperature170°C, initial oven temperature 45
injector was 4.5 minutes, and the splitter was
°C (held for 5 min), increased to 90°C at a rate
opened after 3 minutes. Determinations were
ofperformed
5°C/min and
for of
2 min
increased to
by held
means
gas then,
chromatography
the(Agilent
final temperature
280°C at detector;
a rate of 30°C/min
GC/MS 6890/5973
HP-5, 30
where
it
was
held
for
1
min
.Helium
was
used
as
m × 0.25 mm × 1 µm). The temperatures
were
carrier
gas, atinjector
flow rate
of 1 mL/min.
limits
as follows:
temperature
170°The
C, initial
ofoven
quantification
(LOQ)
values
for
all
compounds
temperature 45° C (held for 5 minutes),
were
0.005 ng/mL.
increased
to 90° C at a rate of 5° C/minutes and
held for 2 minutes then, increased to the final
Statistical
analysis
temperature
280° C at a rate of 30° C/minutes
Mean
concentration
of Helium
Chloroform,
whereurinary
it was held
for 1-minute.
was
used as carrier and
gas, carbon
at flow tetrachloride
rate of 1
Dichloromethane
ml/minutes.
The limits
of quantification
values
between
two groups
(laboratory
technicians
and
for
all
compounds
were
0.005
ng/ml.
Control group) were analyzed and because the
Mean urinary
of chloroform,
distribution
of data concentration
was not normal,
the analysis
DCM
andoutCTC
between
two statistical
groups
was
carried
by means
of two
(laboratory technicians and control group)
procedures: analysis of variance (one- way
were analyzed and because the distribution of
ANOVA)
by Scheff’s
post was
hoc test
and
data wasfollowed
not normal,
the analysis
carried
Kruskal-Wallis
Results
were expressed
as
out by meanstest.
of two
statistical
procedures:
mean
±
S.D.
and
95%
confidence
intervals.
The
analysis of variance (one-way ANOVA)
level
of significance
was set
to 0.05 test
and Pand
followed
by Scheff’s
post-hoc
values
Kruskal–Wallis
test.toResults
were expressed as
>0.05
were assumed
be non-significant.
mean ± standard deviation and 95%
confidenceAND
intervals.
The level of significance
RESULTS
DISCUSSION
was set to 0.050 and P > 0.050 were assumed
Tables
and 2 show the results of the mean urinary
to be1non-significant.
concentration of chloroform, dichloromethane
and
carbon tetrachloride
determinations (ng/mL)
RESULTS
AND DISCUSSION
inTables
the two
groups
of workers
in two
different
1 and
2 show
the results
of the
mean
samples
collected:
before
starting
work
and
the
urinary concentration of chloroform, DCMatand
end
of the
shift.
CTC
determinations
(ng/ml) in the two groups
of workers in two different samples collected:
before starting work and at the end of the shift.
Table 1. Mean urinary concentration (S.D.) of Chloroform, Dichloromethane and carbon tetrachloride in
Table 1: Mean urinary concentration (SD) of chloroform, dichloromethane and carbon tetrachloride in two groups
two groups (n = 40) before starting work.
(n = 40) before starting work
Groups
Chloroform (ng/ml)
Dichloromethane (ng/ml)
Carbon tetrachloride (ng/ml)
Laboratory technicians
0.62 (0.43)
1.15 (1.25)
0.09 (0.08)
Control group members
0.12 (0.21)
0.79 (0.09)
0.03 (0.08)
P
value*
0.024
0.048
0.325
Pvalue
P
value**
0.000
0.000
0.000
Pvalue
*One-way ANOVA, **Kruskal–Wallis test. SD: Standard deviation
Table 2: Mean urinary concentration (SD) of chloroform, dichloromethane and carbon tetrachloride in two groups
(n = 40) at the end of work shift
http://japh.tums.ac.ir
Groups
Chloroform (ng/ml)
Dichloromethane (ng/ml) Carbon tetrachloride (ng/ml)
Laboratory technicians
2.55 (2.10)
1.89 (2.91)
0.09 (0.07)
Control group members
0.23 (0.38)
0.097 (0.11)
0.03 (0.08)
P value*
0.010
0.031
0.431
Groups
Chloroform (ng/ml)
Dichloromethane (ng/ml)
Carbon tetrachloride (ng/ml)
Laboratory technicians
0.62 (0.43)
1.15 (1.25)
0.09 (0.08)
Control group members
0.12 (0.21)
0.79 (0.09)
0.03 (0.08)
P value*
0.024
0.048
0.325
0.000et al., Determination of 0.000
0.000
R. Ahmadkhaniha
urinary concentrations of …
24P value**
*One-way ANOVA, **Kruskal–Wallis test. SD: Standard deviation
Table
2. Mean
urinary
concentration
of Chloroform,
Dichloromethane
and carbon
Tetrachloride
in groups
Table 2:
Mean
urinary
concentration
(SD)(S.D.)
of chloroform,
dichloromethane
and carbon
tetrachloride
in two
two groups
(n
=
40)
at
the
end
of
work
shift.
(n = 40) at the end of work shift
Groups
Chloroform (ng/ml)
Dichloromethane (ng/ml) Carbon tetrachloride (ng/ml)
Laboratory technicians
2.55 (2.10)
1.89 (2.91)
0.09 (0.07)
Control group members
0.23 (0.38)
0.097 (0.11)
0.03 (0.08)
P
value*
0.010
0.031
0.431
Pvalue
P
value**
0.000
0.000
0.000
Pvalue
*One-way ANOVA, **Kruskal–Wallis test. SD: Standard deviation
30 urinary concentrations of all analysts of
The
subjects with job associated with exposure to
solvents (chemistry laboratory technicians)
before starting the work compared with the no
exposure group. The mean concentrations of
chloroform and DCM in chemistry laboratory
technicians were significantly greater than the
control groups. Although the mean levels of CTC
before the work shift in technicians were higher
than the occupationally non-exposed group, a
statistically significant difference could not be
observed (Pvalue= 0.324). The total chloroform
and DCM uptake during the work shift in
laboratory technicians and occupationally nonexposed group were calculated to be on average
1.93±2.1, 0.35±0.6 and 0.69±1.87, 0.02±0.04 ng/
mL, respectively. The concentration of all the
analyets increased during the day for both groups.
The increase is more marked among laboratory
technicians. As can be seen in Table 2, excretion
of chloroform, DCM in laboratory technicians
after the work shift were significantly higher than
the control group (P<0.001). The concentration
of chloroform, DCM in laboratory technicians
increased during the day and reached 2.54±2.1 and
1.84±1.9 respectively. The urine concentration of
chloroform and dichloromethane in both work
shifts followed the order: technicians> control
group members.
The aim of this study was to monitor the degree
of chronic and acute exposure to chlorinated
solvents (chloroform, dichloromethane and
carbon tetrachloride) of two groups of laboratory
technicians and control group members, through
the evaluation of urinary concentrations.
The chemistry laboratory technicians were
chosen for the study because their exposure
was expected to be relatively high and there
http://japh.tums.ac.ir
Airthe
Pollut
& Health
were few reports in literature Jon
biological
monitoring of coincident chlorinated solvents
in urine samples from this group. Regarding
the choice of biomarkers (chloroform, carbon
tetrachloride, dichloromethane) some previous
studies [24, 27, 31, 32] showed a good correlation
between their airborne exposure concentrations
and the biological results. In general, the
advantage of using unmetabolized compounds
as biomarkers is that the urinary concentration
of the unmetabolized substance is less influenced
by inter individual metabolic differences
than the urinary disposition of corresponding
metabolites [33]. For the assessment of exposure
to chlorinated solvents the urinary concentrations
of analytes (chloroform, CTC and DCM) after
the work shift can be used as an indicator of a
day’s (acute) exposure, whereas the urinary
concentration of solvents before the one day
shift allows the monitoring of repeated (chronic)
exposure. A significant difference was observed
in chloroform and DCM concentrations in both
pre and post-shift samples among job categories
(P<0.05 by ANOVA and Kruskal-Wallis test).
The mean chloroform and DCM concentrations in
exposed workers were significantly greater than
unexposed group. Since chloroform and DCM are
widely consumed in the chemistry laboratories,
usually occur at higher concentrations than CTC
(EPA’s Toxic Release Inventory (TRI) database,
2008).
Since there might be a significant risk of dermal
exposure, the laboratory technicians were
requested to wear plastic gloves during the work
shift, so no significant skin exposure occurred
during the study. The CTC level detected in this
study was much lower than levels which are
reported to photocopy centers workers [31]. In
Journal of Air Pollution and Health (Winter 2016); 1(1): 21-26
another study urinary concentration of DCM in
the unexposed general population (120 subjects;
mean age, 38.6±6.6 years) conducted; their
results showed the uptake of small amounts of
DCM [34]. The result of this study showed that
the laboratory technicians are one of the most
exposed groups among occupationally exposed
people. Inhalation is presumably the main route
of exposure in chemistry laboratory technicians.
CONCLUSIONS
The result of this study showed that the laboratory
technicians are one of the most exposed groups
among occupationally exposed people. Inhalation
is presumably the main route of exposure in
chemistry laboratory technicians.
FINANCIAL SUPPORTS
This research has been supported by Tehran
University of Medical Sciences grant (project
No. 86-03-46-6038). Thus, the authors would like
to thank Tehran University of Medical Sciences
for financial support of this study.
COMPETING INTERESTS
The authors declare that they have no conflicts of
interest. ACKNOWLEDGEMENTS
The authors would like to thank Institute
for Environmental Research (IER), Tehran
University of Medical Sciences for cooperation.
ETHICAL CONSIDERATIONS
The authors state that they have no ethical
considerations.
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